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Photochemical and Non-Photochemical Quenching of Chlorophyll Fluorescence Induced by Hydrogen Peroxide

Christian Neubauer and Ulrich SchreiberLehrstuhl Botanik I, Universität Würzburg, Mittlerer Dallenbergweg 64,D-8700 Würzburg, Bundesrepublik Deutschland

Z. Naturforsch. 44c, 262—270 (1989); received Decem ber 12, 1988

Chlorophyll Fluorescence. Fluorescence Quenching, Hydrogen Peroxide. Active Oxygen. Ascorbate Peroxidase

Chlorophyll fluorescence quenching induced by H 20 2 in intact spinach chloroplasts was investi­gated with a modulation fluorometer which allows to distinguish between photochemical and non­photochemical quenching com ponents by the so-called saturation pulse method. Residual catalase activity was removed by washing and percoll gradient centrifugation. H 20 2 was found to induce pronounced photochemical and non-photochemical quenching, characteristic for the action of a Hill reagent, with a half-maximal rate already observed at 5 x 10~6 m . The saturation characteris­tics and maximal rate o f H 20 2-reduction were very similar to those of methylviologen reduction. H 20 2-dependent quenching was stimulated by ascorbate and inhibited by cyanide and azide in agreement with previous findings by other researchers that H 20 2-reduction involves the ascorbate peroxidase scavenging system and that the actual “Hill acceptor” is an oxidation product of ascorbate, i.e. monodehydroascorbate or dehydroascorbate. With well-coupled intact chloro­plasts reducing C 0 2 at 150 (irnol (mg C hl)“ 'h_ l, iodoacetamide stopped CO;-dependent 0 2- evolution and consequent addition of 10“3 m H 20 2 produced an 0 2-evolution rate o f 240 (.irnol (mg C hl)“ 'h_ l. It is concluded that light-dependent H20 2 reduction is a very efficient reaction in intact chloroplasts. As H 20 2 formation and consequent reduction also occur in vivo, the corre­sponding quenching should be considered when assimilatory electron flow is estimated from quenching coefficients. It is suggested that proton flux associated with H20 2-formation and reduc­tion may be important for the adjustment of appropriate A T P/N A D PH ratios required for C 0 2- fixation in vivo. Furthermore, H 20 2-reduction may serve as a valve reaction whenever Calvin cycle activity is limited by factors different from N A D PH supply, thus protecting against photo- inhibitory damage.

Introduction

Chlorophyll fluorescence can give information on almost all aspects of photosynthesis in isolated chloroplasts as well as in intact leaves (for reviews, see ref. [1—4]). Fluorescence yield in vivo is lowered by two fundamentally different mechanisms, leading to photochemical and non-photochemical quench­ing. Photochemical quenching is caused by charge separation at PS II reaction centers, while non­photochemical quenching may be due to a number of other non-radiative de-excitation processes in PS II. Most of non-photochemical quenching is linked to the internal acidification of the thylakoids and is therefore termed “energy-dependent” quenching [5].

Abbreviations: PS, photosystem; D C M U , 3-(3,4-dichloro- phenyl-)l ,1-dimethylurea; qP, coefficient o f photochemical quenching; g NP, coefficient o f non-photochemical quenching.

Reprint requests to Dr. U. Schreiber.

Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen0341 - 0382/89/0300 - 0262 S 0 1 .30/0

Photochemical and non-photochemical quenching components can be determined by the so-called sat­uration pulse method [6—9], the practical application of which became possible with the introduction of a selective modulation fluorometer [9, 10].

Although photochemical quenching may be con­sidered a reliable measure of PS II charge separation rate, it has been pointed out before that the rate will not necessarily reflect overall assimilatory rate [4], In previous work, we have considered electron flow to 0 2 [4, 11] and cyclic flow around PS II [11 — 14] to cause “non-assimilatory” photochemical quenching.

0 2-Reduction is an inevitable consequence of aerobic photosynthesis, leading to formation of superoxide, H 2 0 2 and other active oxygen species (for reviews, see ref. [15, 16]). The scavenging of active oxygen is of utmost importance for chloroplast protection against oxidative damage. For example, H 2 0 2-concentrations as low as 10“'' m were found to cause deactivation of Calvin cycle enzymes [17, 18]. Extensive work by Asada and coworkers [16, 19—21] and other researchers [22—24] has established that

Ch. Neubauer and U. Schreiber • Fluorescence Quenching by Hydrogen Peroxide 263

chloroplasts not only contain several types of superoxide dismutase [25, 26] but also a very effec­tive H 2 0 2-scavenging system, the light-dependent as- corbate peroxidase. Catalase appears to be restricted to the peroxisomes [19, 21, 24, 25],

The present investigation aims at the question to what extent fluorescence quenching is effected by0 2-dependent electron flow, which comprises reduc­tion of 0 2 and of the consequently formed H 2 0 2. In this first report we will describe the effect of exter­nally added H 2 0 2 on fluorescence quenching. It will be shown that the light-driven reduction of H 2 0 2 via the ascorbate peroxidase is very efficient, matching the reduction of the well known Hill reagent methyl- viologen. In a following communication [27] the in­vestigation will be extended to the effect of endogen­ously formed H 2 0 2.

Materials and Methods

Intact chloroplasts were isolated from spinach essentially following the method of Jensen and Bassham [28], as modified by Egneus et al. [29]. These chloroplasts were further purified by percoll gradient centrifugation, as described by Nakano and Asada [19], to remove residual catalase activity and to further increase the proportion of chloroplasts with intact envelopes. The degree of intactness amounted to 90-95% , as judged by the ferricyanide method [30]. Catalase activity of the final chloroplast preparation was very low, as evidenced by the fact that upon addition of 4 ^mol H 2 0 2 to a chloroplast suspension containing 150 nmol chlorophyll not more than 1 0 nmol 0 2 was spontaneously formed in the dark. When catalase was added externally, its concentration was 3000 units ml-1. Chloroplasts were suspended isotonically in a reaction medium contain­ing 330 m M sorbitol, 50 m M K-Tricine pH 7.6, 1 m M

MgCl2, 0.25 mM Na2 P 0 4 and 2 mM N aH C 03. If not stated otherwise, 10 m M Na-ascorbate was present to counteract efflux of intrinsic ascorbate into the medium. In presence of ascorbate the intact chloro­plasts displayed high-saturated rates of C 0 2 -depend- ent 0 2-evolution ranging between 130 and 160 [omol (mg Chl)_1h_1. Class D chloroplasts were prepared from intact chloroplasts by 30 s exposure to a hypo­tonic buffer containing 10 m M MgCl2, 10 m M Na-as- corbate and 5 m M K-Tricine pH 7.6, followed by isotonic resuspension by addition of an equal amount of a medium containing the same additions as the

medium for intact chloroplasts but at double con­centrations. H 2 0 2-Solutions of 1 m to 10- 1 m were freshly prepared every day from a 30% stock solu­tion (Merck). Small aliquots were kept on ice in polypropylene caps each of which was used for one experiment only, to minimize a lowering of effective H 2 0 2-concentration by spontaneous decomposition. If not stated otherwise, tem perature during measure­ments was 18 °C and chlorophyll concentration was 60 ng ml-1.

Chlorophyll fluorescence and oxygen evolution were measured as described before [1 1 ], with a mod­ulation fluorometer (PAM system, H. Walz, Effel­trich, F .R .G .) equipped with fiber optics and suit­able adaptors for a laboratory-built cuvette, contain­ing a side-port Pt/Ag/AgCl electrode. Actinic light was obtained from a tungsten halogen lamp, with a broad red band (about 630—700 nm) selected by RG 630 (Schott) and DT Cyan (Balzers) filters. Repetitive pulses of saturating heat-filtered white light were applied with the help of a relais-controlled fiber illuminator (FL 103, triggered by PAM 103, Walz). Pulse length was 800 ms and pulse intensity 2000 W -m ~2. The intensity of the modulated meas­uring beam was very low (about 1 0 - 2 W -m -2) when the quasi-dark level of fluorescence yield, F0, was measured at 1.6 kHz modulation frequency. Simul­taneously with the onset of actinic illumination, mod­ulation frequency was automatically switched to 100 kHz by appropriate circuitry in the PAM 102 unit, in order to improve the signal/noise ratio for the kinetic recordings. If not stated otherwise, actinic light intensity was 20 W -m -2, as provided at the maximal setting of the light-emitting diode source (102 L) provided with the PAM fluorometer.

Results and Interpretation

In Fig. 1 the basic effect of H 2 0 2-induced fluores­cence quenching is demonstrated with intact spinach chloroplasts. This example also may serve to illus­trate the separation of photochemical and non­photochemical quenching components by the satura­tion pulse method. When the weak modulated meas­uring beam is switched on, the quasi-dark level fluorescence yield, Fo, is monitored. Upon applica­tion of a pulse of saturating light the dark-adapted sample displays the maximal fluorescence yield, Fm. F0 and Fm represent extreme values of fluorescence yield, when all PS II reaction centers are open or

264 Ch. Neubauer and U . Schreiber • Fluorescence Quenching by Hydrogen Peroxide

( 0 . 1 3 )

‘5x10

HnO2U2catalase

5x10 M

DCMU

Fig. 1. H :0 :-induced fluorescence quenching in coupled intact spinach chloroplasts. F„, dark-level fluorescence yield monitored by ex­tremely weak modulated measuring beam. Fm, maximal fluorescence yield of dark-adapted sample, determined by a pulse of saturating light. Continuous actinic light of 20 W ■ m 2 was turned on at the open arrow. Saturation pulses were applied repetitively every 20 s aft­er onset of continuous illumination. Saturated fluorescence yield, Fs, observed during these pulses is variable, being lowered with respect to Fm by non-photochemical quenching. Photo­chemical quenching is reflected by the relative amplitude of the spikes produced during a sat­uration pulse. Some values of the photochem i­cal quenching coefficient, q P (calculated ac­cording to ref. [9]) are written in brackets above the corresponding part of the induction curve.

closed, respectively, without any non-photochemical quenching being induced. When continuous actinic light is switched on, fluorescence yield first rapidly increases and then slowly declines again (Kautsky effect). By repetitive application of saturating light pulses it can be distinguished to what extent the slow fluorescence changes are due to changes in photo­chemical or in non-photochemical quenching. The lowering of the saturation pulse fluorescence yield, Fs, with respect to Fm is expression of non-photo- chemical quenching. That part of quenching which can be eliminated by saturation pulses (i.e. rep­resented by the “spikes”) is due to photochemical quenching (for a detailed definition of the coeffi­cients of photochemical quenching, q ? or qQ, and of non-photochemical quenching, qNP or qE, see e.g. ref. [4, 8 , 9]). When H 2 0 2 is added to the stirred chloroplast suspension, there is a large, rapid drop of fluorescence yield followed by a smaller and slower decline. The rapid drop is caused primarily by an increase in photochemical quenching, while the slower decline is paralleled by an increase in non­photochemical quenching and by a small decrease in photochemical quenching. When catalase is added to decompose H 2 0 2, photochemical quenching returns almost to its value before H 2 0 2 addition while there is only a minor reversion of non-photochemical quenching. Photochemical and most of non-photo­chemical quenching are effectively eliminated by DCMU, which is known to block electron transfer at the PS II acceptor side.

The rapid increase of fluorescence upon onset of actinic illumination specifically reflects changes in photochemical quenching, as the development of non-photochemical quenching is relatively slow [6—9]. With proper time resolution, this fluorescence increase displays two phases, yielding the so-calledO-I-P transient. Fig. 2 shows the effect of H 20 2, ap­plied at different concentrations to dark-adapted chloroplasts, when the O-I-P transient was induced 5 s following H 2 0 2-addition. In Fig. 3 the charac­teristic fluorescence levels O, I and P are plotted in dependence of the applied H 2 0 2-concentration. It is apparent that H 2 0 2 affects only the P-level, while O- and I-levels are unaffected. Half-maximal P-level suppression is observed at about 5 x 1CT6 m .

The changes in fluorescence characteristics by H 2 0 2, as depicted in Fig. 1—3, are typical for the action of Hill reagents, which prevent accumulation of reduced plastoquinone between the two photosys­tems. According to extensive studies by Asada and co-workers [19—21] and by other researchers [22—24], light-driven H 20 2-reduction involves the action of the ascorbate peroxidase scavenging system present in chloroplasts. In Fig. 4 the essential role of ascorbate in the H 2 0 2-induced quenching reaction is demonstrated for osmotically ruptured chloroplasts, with the intrinsic ascorbate being diluted into the reaction medium. In this experiment the formation of a proton gradient and consequent development of energy-dependent fluorescence quenching was pre­vented by presence of the protonophore nigericin.

Ch. Neubauer and U. Schreiber • Fluorescence Quenching by Hydrogen Peroxide 265

P

Time

Fig. 2. Rapid rise kinetics of chlorophyll fluorescence in dependence of the H20 2-concentration. H 20 2 was added 5 s before recording of the induction curves. The characteristic fluorescence levels O (dark). I (intermediate) and P (peak) are indicated. Actinic light intensity, 20 W -m 2.

Chloroplasts without externally added ascorbate dis­played only weak and transient photochemical quenching by H 2 0 2 (Fig. 4 A). When 5 mM ascorbate was present (during osmotic rupture and the conse­quent fluorescence experiment) addition of H 2 0 2

caused pronounced photochemical quenching which persisted as long as H 2 0 2 was available (Fig. 4B).

^(^-Concentration, mol'l 1

Fig. 3. Dependence of the characteristic fluorescence levels in a rapid induction curve on the H 20 2-concentration. For conditions, see Fig. 2.

There was an abrupt reversal of quenching when H 2 0 2 was consumed. Quenching lasted about two times longer when a twofold amount of H 2 0 2 was added. As expected, catalase reversed photochemi­cal quenching by rapid decomposition of the H 2 0 2.

Fl2 0 2-induced fluorescence quenching is suppress­ed by cyanide and azide, but not by iodoacetamide, in agreement with previous reports showing that cyanide and azide, but not iodoacetamide, inhibit chloroplast peroxidase activity [19—21]. Fig. 5 shows that in presence of 1 mM KCN H 2 0 2-induced quench­ing is almost completely prevented. The same con­centration of KCN had no effect on the quenching induced by methylviologen, as shown in Fig. 6 . It may be concluded that at the given concentration, the cyanide did not yet affect electron transport at the level of plastocyanine. Hence, the results of Fig. 5 emphasize that H 2 0 2 is not directly reduced by the electron transport chain but rather via the ascorbate peroxidase system [20, 21]. Accordingly, the actual “Hill acceptor” must be an oxidation product of ascorbate, i.e. monodehydroascorbate or dehydro- ascorbate.

In first approximation, photochemical fluores­cence quenching may be considered a relative meas­ure of the rate of charge separation at PS II and, consequently, in the steady state of overall electron flow rate [1—4, 31], We have compared the photo­chemical quenching induced by H 2 Q 2 with that

266 Ch. Neubauer and U. Schreiber ■ Fluorescence Quenching by Hydrogen Peroxide

no ascorbate added + 5 mM ascorbate

/KWUuJUJ

catalase

l-l min -I catalase |

ft 1'5x10 4M t 10_3Mi"2xl0"3M

IT

CSIo

| h 2°2 h2o25x10 M

h 2o 2

Fig. 4. Stimulation of H :O r induced fluorescence quenching by ascorbate. Class D chloroplasts in the presence of 10 m

nigericin. Chemical additions are indicated at the corresponding arrows.

Fig. 5. Inhibition of H 20 2-dependent fluorescence quenching by cyanide. Intact chloroplasts in presence of 10 m

nigericin. Other conditions as for Fig. 1.

5xl0'5M

MV catalase

_S*J1 m M KCN

5x10 M

MV

IT

catalase

Fig. 6. M ethylviologen-induced quenching of chlorophyll fluorescence. Conditions as for Fig. 5. Experiment showing that cyanide at given concentration does not affect m ethylviologen-dependent electron transport.

Ch. Neubauer and U. Schreiber • Fluorescence Quenching by Hydrogen Peroxide 267

100 200 300

Light intensity, W nf2

400 500

Fig. 7. Light-intensity dependence of the photochemical fluorescence quenching induced by addition of 1CT3 m H 20 2 or 10“3 m methylviolo- gen to intact, uncoupled spinach chloroplasts. Quenching was deter­mined under the conditions of the experiments in Fig. 5 and 6, except that different intensities of red ac­tinic light were applied (see M ate­rials and M ethods). Presence of 10-7 m nigericin. In the experiment with m ethylviologen, 10 3 m KCN was added to stabilize the formed H-iO t.

caused by methylviologen, at a variety of light inten­sities. To obtain maximal, uncoupled rates, the pro- tonophore nigericin was added. In the methylviolo­gen experiment, 1 m M KCN was added to suppress any residual catalase activity and to inhibit ascorbate peroxidase. The results are shown in Fig. 7. It is apparent that H 2 0 2, in combination with the ascor­bate peroxidase system, is capable of supporting as high or even somewhat higher rates of electron trans­port as the well-known and efficient Hill reagent

methylviologen. Under the conditions of the experi­ment of Fig. 7 methylviologen-dependent 0 2-uptake saturated at a rate of about 700 fimol 0 2

(mg Chl)- 1h-1. The corresponding light intensity de­pendence is shown in Fig. 8 . These findings confirm previous conclusions by other researchers [19—21, 24], that the potential rate of H 20 2-scavenging within the chloroplast is very high. As long as this scaveng­ing system is intact, its rate should exceed by far that of any possible formation of H 2 0 2.

Light Intensity, W m'2

Fig. 8. Light-intensity dependence of oxygen up­take in presence o f 1 0 3 m m ethylviologen, 1 0 7 m

nigericin and 10~3 m KCN. Conditions as in Fig. 7. Data points from two independent experiments are presented.

268 Ch. Neubauer and U. Schreiber • Fluorescence Quenching by Hydrogen Peroxide

Under coupled conditions, a direct comparison of C 0 2-dependent 0 2-evolution and 0 2-evolution link­ed to reduction of externally added H 2 0 2 is possible. Fig. 9 shows simultaneous recordings of 0 2-evolution and fluorescence quenching. 2 0 mM ascorbate was present to provide optimal conditions for the peroxi­dase. A non-saturating light intensity was chosen to allow an appreciable level of photochemical quench­ing. The C 0 2-dependent rate amounted to 60 jimol0 2 (mg Chl)- 1h-1, paralleled by q P = 0.29. When C 0 2-fixation was inhibited by iodoacetamide, 0 2- evolution was suppressed and qP decreased to 0.23. This decrease in photochemical quenching is surpris­ingly small, suggesting that electron flow to non-as- similatory acceptors was almost matching C 0 2 -de- pendent flow, once this was inhibited. Actually, it is important to note, that non-photochemical quench­ing was practically not affected by the inhibition of Calvin cycle. When 10~ 3 m H 2 0 2 was added, there was practically no spontaneous 0 2-formation, show­ing that with the given chloroplast preparation re­sidual catalase activity was negligibly small. There was light-dependent O2*evolution which ceased again, when H 2 0 2 was consumed. H 2 0 2-dependent rate was 240 |amol 0 2 (mg Chl)- 1h_1, accompanied by q ? = 0.50. Hence, in this particular experiment

0 2-evolution linked to reduction of externally added H 20 2, exceeded C 0 2-dependent 0 2-evolution (as measured before iodoacetamide addition) by a factor of four. It is remarkable that such a high electron flow rate is possible under coupled conditions when there is no ATP-requirement.

Discussion and Conclusion

Light-dependent reduction of hydrogen peroxide by intact spinach chloroplasts has been previously described by several researchers [19—21, 24], In par­ticular, the extensive work of Asada and co-workers (for a recent review, see ref. [16]) has emphasized the essential role of the ascorbate peroxidase in the chloroplast stroma for a highly efficient scavenging of H 2 0 2. This earlier work was based on measure­ments of 0 2 -evolution, 0 2-uptake and H 20 2 -con- sumption by polarography and mass spectroscopy. The main purpose of the present contribution was to investigate in what way chlorophyll fluorescence quenching is influenced by the H 2 0 2-scavenging sys­tem. Not unexpectedly, it was found that H 2 0 2 add­ed to intact chloroplasts causes substantial photo­chemical and non-photochemical quenching. The properties of this H 2 0 2-induced quenching are in ex-

0.7

0.6

0.5

0.4

0.3

Fig. 9. Simultaneous recordings o f modulated chlorophyll fluorescence and polarographically measured oxygen concentration. Effect of iodo­acetamide to suppress C 0 2-dependent 02-evolu­tion. and stimulation of 0 2-evolution and photo­chemical quenching by addition of H :0 : . Actinic light intensity. 150 W • m 2. Temperature. 20 °C. Chlorophyll concentration. 40 ng m l"1.

Ch. Neubauer and U . Schreiber • Fluorescence Quenching by Hydrogen Peroxide 269

cellent agreement with the H 2 0 2-scavenging mecha­nism elucidated by previous work:1) Quenching is stimulated by ascorbate, confirming

that ascorbate plays an essential role.2) Cyanide and azide inhibit quenching, suggesting

that H 2 0 2 is not directly reduced by the photo­synthetic electron transport chain, and that the as­corbate peroxidase is involved.

3) Very low H ?02-concentrations are required for stimulation of photochemical quenching. Actual­ly, the concentration for 50% stimulation of5 x 10- 6 m is lower than the previously estimated value of 25 x 10- 6 m [24], presumably because the fluorescence response can be more readily meas­ured than 0 2-evolution when small amounts of H 2 0 2 are rapidly consumed in the light.

4) Substantial photochemical quenching is induced by H 2 0 2 even at very high light intensities, show­ing that the capacity of the H 2 0 2-scavenging sys­tem is very high. Maximal rates of H 2 0 2-reduction match that of methylviologen reduction. Hence, H 2 0 2 may be considered a very effective naturally occurring Hill reagent.

These results appear significant for three main reasons:

First, it has been dem onstrated that chlorophyll fluorescence, in particular by application of the sat­uration pulse method, allows insights into the H 2 0 2- scavenging mechanism of the chloroplasts. This method can also be used with intact leaves, and in several respects its application may be advantageous to that of polarography and mass spectroscopy. Knowledge about H 2 0 2-formation and scavenging is particularly important for an assessment of chloro­plast performance under stress conditions when elec­tron consumption in the Calvin cycle is suppressed. H 2 0 2-reduction could serve as a valve reaction, pre­venting photoinhibitory damage of the chloroplasts whenever Calvin cycle is limited by factors different form NADPH. For this function it may be important that H 2 O r dependent electron flow can display very high rates even under coupled conditions.

Second, the finding of strong quenching induced by small amounts of externally added H 2 0 2 raises the question of the possible contribution of such H 2 Q2-

dependent quenching also in intact leaves under in vivo conditions. Univalent reduction of 0 2 to superoxide and following dismutation to H 2 0 2 and0 2 may be considered an inevitable consequence of photosynthetic electron flow and the efficient scavenging of H 2 0 2 is essential for plant survival, as very low H 2 0 2-concentrations cause severe inhibition of stroma enzymes [17, 18, 20], W henever H 2 0 2-for- mation and scavenging occur in a leaf, this is equiva­lent to the introduction of a Hill reagent, like methyl­viologen, with the effect of appreciable fluorescence quenching. Photochemical and non-photochemical quenching produced in this way would overlap quenching related to assimilatory electron flow, and, hence, the determination of this flow would be com­plicated. In a following communication, we will deal with this aspect in more detail [27],

Third, if H 2 0 2-formation indeed is an inevitable consequence of aerobic photosynthetic electron transport, then this is also true for the vectorial pro­ton flux linked to formation and scavenging of H 2 0 2. The resulting proton motive force will contribute to ATP-synthesis, and this contribution may be impor­tant for the establishment of an overall ATP/2e- ratio of 1.5 required for C 0 2-fixation in C3-plants. Linear electron flow to NADP is believed to provide an ATP/2e_ of 1.33 and it is a m atter of controversy whether additional ATP-formation is primarily due to pseudocyclic or cyclic phosphorylation [29, 32, 33] or linked to a Q-cycle involving the Cyt b /f complex [34]. W hether H 2 0 2-reduction carries sufficient flux in vivo to be of any relevance for ATP-synthesis can not be decided on the basis of the above data. This question will be addressed in a following publication[27].

Acknowledgements

We wish to thank K. Asada for stimulating discus­sions on the role of active oxygen in photosynthesis, which were very helpful for the interpretation of our fluorescence data. Thanks are also due to U. Heber and W. Kaiser for many helpful suggestions. A Weber is thanked for technical assistance. This work was supported by the Deutsche Forschungsgemein­schaft, SFB 176 and La 54/32.

270 Ch. Neubauer and U. Schreiber • Fluorescence Quenching by Hydrogen Peroxide

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